1. Field of the Invention
Beginning with the observation by B. S. Blumberg et al. in 1961 of a serological precipitin reaction between the blood serum of a multiply transfused patient and blood serum from an Australian aborigine, it has been established that an antigen exists which is commonly associated with hepatitis. See B. S. Blumberg et al. Nat. Acad. Med. 44:112, 1566, (1968) and H. S. Alter and B. S. Blumberg, Blood, 27:3, 297 (1966) and A. J. Zuckerman, Nature, 223:569 (1969). Originally known as the Australia antigen, this material is now generally referred to in the literature simply as the hepatitis associated antigen (HAA).
Hepatitis is clinically divided into two general categories, i.e., serum hepatitis, usually termed HBAg or type B hepatitis, and infectious hepatitis which is usually termed type A hepatitis. Serum hepatitis (type B) exists in at least two subtypes, i.e., a-d and a-y. The current available means of detection of hepatitis are primarily directed toward serum hepatitis (HBAg or type B) and are based on the serological reaction of the antigen associated with the disease process. The antigen involved in this invention will be referred to hereinafter simply as HAA, but includes the high molecular weight protein associated with viral infection of the liver. This antigen (HAA) is extremely rare in the blood of the normal American population and has been reported to occur to an extent of less than 0.1%. The HAA antigen has been detected in patients suffering from acute myelogenous leukemia, chronic lymphocytic leukemia, and acute lymphocytic leukemia. The antigen (HAA) has also been detected in institutionalized patients with Down's syndrome who have a tendency to exhibit sub-acute hepatitis as reported by Krugman and others in the New England Journal of Medicine, 281:119-122, (1969). The HAA antigen, as indicated by its name, has been found to have a very high incidence in patients who have suffered infectious hepatitis. Inasmuch as a very large number of patients receiving transfusions of blood materials contract hepatitis due to the origin of the transfused blood materials, much work has been done in recent years and is still being done to reduce infection from this source. Much of this work has centered around schemes to detect the presence of the hepatitis associated antigen (HAA) in blood prior to administration of the blood or blood materials to the patient. Canadian Medical Association Journal, 106 Special Issue on Viral Hepatitis, February 26, 1972; Austrailia Antigen and Hepatitis, B. S. Blumberg, A. I. Sutnick, W. T. London and I. Millman, C.R.C. Press, Cleveland, Ohio, (1972).
2. The Prior Art
In general, the serological schemes previously proposed for the detection of HAA in blood depend upon the isoprecipitin reaction of antibody-containing serum, commonly found in multiply-transfused patients and currently produced in animals immunized with HAA, with the antigen (HAA) found most frequently in the serum of patients with hepatitis, or a lesser extent in the other disorders referred as above. Two well-known methods of this type are immunodiffusion (ID) and complement fixation (CF). Both of these methods require a minimum eight hour incubation period and are not, therefore, suitable as rapid screening techniques in blood banks. An electrophoretically enhanced diffusion; i.e., immunoelectroosmophoresis (IEOP) or counter-electrophoresis (CEP) has been recently developed by Prince and Burke (Scienes 169:593 (1970)). This method is very sensitive and can be carried out in about 30 minutes.
S. F. Malin and J. R. Edwards of Villanova University (Nature, New Biology 235 182 (1972)) have recently developed a latex inhibition assay (LAT) for HAA in which latex particles are employed as the indicator system. The agglutination of the particles indicates the presence of HAA. The assay is simple and rapid.
The American Association of Blood Banks has recommended that the presence or absence of HAA should be established as one of the criteria for the administration of blood and blood products prior to their administration. The currently accepted method for this purpose is RIA, but this stand has been strongly questioned in (Blood 43, 947, (1973)) and N. Eng. J. Med. 289, 385 (1973), due to its expense and the fact that blood shown to be negative by RIA has caused post-transfusion hepatitis. The radioimmuno assay (RIA), employs a radioactively labeled protein which combines with the HAA to give a radioactive antibody-antigen complex incorporating a radioisotope in an amount proportionate to the amount of HAA in the serum.
Inasmuch as whole blood and blood materials have a relatively short shelf life, there is a need for a rapid method for screening blood for the presence of HAA prior to administration. A truly rapid test would also have obvious utility in blood banks, since a potential donor's blood could be sampled and tested prior to withdrawing the main blood donation, thus saving the time and expense of collecting quantities of unusable blood.
A further disadvantage of previously proposed methods for testing for hepatitis is that HAA is the only hepatitis related antigen or protein which is indicated by the tests using hepatitis associated antibody. The specificity of the antibody-antigen reaction precludes reactions with dissimliar, but possibly related, antigens. Probably a major portion of the blood and blood products capable of transmitting hepatitis contain HAA as the causative agent or associated with the causative agent. However, it is possible that numerous other antigens or other high molecular weight proteins having a similarity to HAA may indicate and/or be capable of transmitting hepatitis. Such other antigens or proteins are not detected by previously known tests for hepatitis.
It is the primary object of the present invention therefore, to provide a rapid test for detecting the presence of HAA in biological materials and especially those obtained from the human body, particularly blood and blood materials.
It is another object of the invention to provide an improved test for detecting not only HAA, but also other agents in blood materials which cause or are associated with hepatitis, and which is simple enough to be carried out routinely by persons without specialized technical training.